Australian Centre for Neutron Scattering, Australian Nuclear Science and Technology Organisation (ANSTO), Lucas Heights, New South Wales 2234, Australia.
Langmuir. 2017 Oct 31;33(43):12497-12509. doi: 10.1021/acs.langmuir.7b02872. Epub 2017 Oct 20.
CLIC1 belongs to the ubiquitous family of chloride intracellular ion channel proteins that are evolutionarily conserved across species. The CLICs are unusual in that they exist mainly as soluble proteins but possess the intriguing property of spontaneous conversion from the soluble to an integral membrane-bound form. This conversion is regulated by the membrane lipid composition, especially by cholesterol, together with external factors such as oxidation and pH. However, the precise physiological mechanism regulating CLIC1 membrane insertion is currently unknown. In this study, X-ray and neutron reflectivity experiments were performed to study the interaction of CLIC1 with different phospholipid monolayers prepared using POPC, POPE, or POPS with and without cholesterol in order to better understand the regulatory role of cholesterol in CLIC1 membrane insertion. Our findings demonstrate for the first time two different structural orientations of CLIC1 within phospholipid monolayers, dependent upon the absence or presence of cholesterol. In phospholipid monolayers devoid of cholesterol, CLIC1 was unable to insert into the lipid acyl chain region. However, in the presence of cholesterol, CLIC1 showed significant insertion within the phospholipid acyl chains occupying an area per protein molecule of 6-7 nm with a total CLIC1 thickness ranging from ∼50 to 56 Å across the entire monolayer. Our data strongly suggests that cholesterol not only facilitates the initial docking or binding of CLIC1 to the membrane but also promotes deeper penetration of CLIC1 into the hydrophobic tails of the lipid monolayer.
CLIC1 属于氯离子细胞内通道蛋白家族，该家族在物种间广泛存在且进化上保守。CLIC 蛋白的独特之处在于它们主要以可溶性蛋白的形式存在，但具有自发从可溶性转化为完整膜结合形式的有趣特性。这种转化受膜脂组成的调节，特别是胆固醇，以及氧化和 pH 等外部因素。然而，目前尚不清楚调节 CLIC1 膜插入的确切生理机制。在这项研究中，我们进行了 X 射线和中子反射实验，以研究 CLIC1 与不同磷脂单层的相互作用，这些单层是使用 POPC、POPE 或 POPS 制备的，并且存在或不存在胆固醇，以便更好地理解胆固醇在 CLIC1 膜插入中的调节作用。我们的研究结果首次证明了 CLIC1 在磷脂单层中有两种不同的结构取向，这取决于胆固醇的存在与否。在没有胆固醇的磷脂单层中，CLIC1 无法插入脂质酰链区。然而，在胆固醇存在的情况下，CLIC1 显示出在磷脂酰链内的显著插入，每个蛋白质分子占据的面积为 6-7nm，整个单层中 CLIC1 的总厚度范围为 50-56Å。我们的数据强烈表明，胆固醇不仅促进了 CLIC1 与膜的初始对接或结合，而且还促进了 CLIC1 更深地渗透到脂质单层的疏水尾部。
